CFTR channel activator compounds and pharmaceutical compositions containing same

ABSTRACT

CFTR channel activator compounds from the benzo[c]quinolizinium family or families of compounds derived therefrom, as well as pharmaceutical compositions containing said compounds, and the uses thereof, particularly for treating cystic fibrosis, are disclosed.

This application is a 371 of PCT/FR97/01436 filed Jul. 31, 1997, now WO98/05642 Feb. 12, 1998.

The present invention relates to CFTR channel activator compounds,pharmaceutical compositions comprising the latter, and their use in thecontext of treatment of pathologies such as cystic fibrosis.

In an epithelial cell, transportation of water and electrolytes isassociated with an increase in the permeability of the membranes to theions K⁺, Na⁺ and Cl⁻. These movements are linked to the activity of ionchannels, that is to say specialized proteins integrated into themembrane allowing passive diffusion of ions. The techniques of molecularelectrophysiology (patch clamping) allow recording at the unit level ofthe openings and closings of an ion channel and make it possible tostudy transepithelial ion transportations their regulation and theirpathological dysregulation.

Among the numerous pathologies associated with the physiology ofepithelial cells, cystic fibrosis is also regarded as a pathology of ionchannels to the extent that the protein involved is a chloride channel,the CFTR channel, meaning “cystic fibrosis transmembrane conductanceregulator”. Mucoviscidosis, or cvstic fibrosis (CF) in Anglo-Saxonterminology, is the most common recessive autosomal genetic disease inCaucasian populations. In the United States and in the majority ofEuropean countries, the incidence of carriers of the CF gene is 1 in 20to 1 in 30. Cystic fibrosis affects the exocrine glands of the humanorganism. The main expression sites of the CFTR protein are the exocrinepancreas, the lungs, the sudoriparous glands, the intestine and thecardiac tissue. The attention paid to this disease has had significantconsequences on understanding the secretory mechanisms of normalepithelial cells. The epithelial cells of the exocrine glands of theintestine, the pancreas or the lungs control transportation of salt andwater into these organs. In cases of cystic fibrosis, mutations of theCF gene alter the properties and function of the CFTR channel. Thetransportation of electrolytes then becomes abnormal and leads tochronic pulmonary obstructive disorders, to pancreatic insufficiency, tobacterial pulmonary conditions, to an abnormally concentratedsudoriparous secretion and to masculine infertility. The defectivesecretion is linked with functioning of selective ion channels forchloride ions (CFTR channels) which are located in the apical membraneof cells and the activity of which is controlled by the cyclic AMProute.

The protein CFTR is a glycoprotein of 1.480 amino acids of molecularweight 170 kD divided into five fields (Riordan et al. 1989), twotransmembrane segments each with 6 alpha-helices (numbered 1 to 12, eachcomprising 21 to 22 amino acids), two nucleotide binding fields (NBF1and 2) and a large hydrophilic regulation field (field R). The proteinCFTR, from its molecular structure, belongs to the family of membranetransporters (ABC meaning ATP-binding cassette).

The ABC transporters constitute a family of membrane proteins which arehighly conserved in evolution. They are involved in the translocation ofvarious substrates through cell membranes. However, while in prokaryotesseveral transporter/substrate pairs have been defined, this informationis rarer in eukaryotes. In mammals, the majority of ABS transporters areassociated with a pathology. The protein CFTR involved in cysticfibrosis, glycoprotein P (MDR: multi-drug resistance) involved in therejection of antitumoral cytotoxic drugs and the protein ABC 1, recentlydescribed as playing an essential role in endocytosis of apoptoticbodies by the macrophage, may be mentioned. CFTR controls thetransportation of transepithelial chloride and hydration of mucouscompartments, while one of the isoforms of MDR is involved in thetranslocation of phosphatidylcholine. These three ABC proteins, whichhave a structure of two times 6 transmembrane segments, have two fieldswhich bind and hydrolyse nucleotides (NBF) and a regulator field. Theregulation of CFTR has been studied in particular.

Two complex processes control the activity of the CFTR channel:phosphorylation of field R by kinase proteins and binding (and perhapshydrolysis) of ATP on the two NBF fields. The dephosphorylation of theCFTR channel causes a loss in activity of the channel up to its closure(Tabcharani et al., 1991, Becq et al. 1993a. Becq et al., 1994). Inaddition, the CFTR channel is associated with a membrane phosphatasewhich controls the activity and state of phosphorylation of the channel(Becq et al., 1993b. Becq et al., 1994).

The gene which codes for the protein CFTR has been isolated by molecularcloning and identified on chromosome 7 (Kerem et al., 1989, Riordan etal., 1989). The identification of the gene and its involvement in cysticfibrosis has been confirmed by the location of a deletion of three basepairs in a coding region (exon 10) of the CF gene originating from CFpatients. This mutation corresponds to the deletion of a phenylalaninein position 508 (ΔF508) of the protein in NBF1. The frequency ofoccurrence of this mutation is 70% on average in the gene analyses (Tsui& Buchwald. 1991). The consequences of this mutation are dramatic, sincethe abnormal protein produced by transcription of the mutated gene(ΔF508) is no longer capable of ensuring its function in thetransportation of chloride of the epithelial cells affected. The absenceof a chlorine current after stimulation of epithelial cells of exocrineglands by cAMP is the main characteristic demonstrating the presence ofan anomaly of the CF gene, and in particular the mutation (ΔF508). Morethan 300 mutations have been identified to date on the CF gene. Thehighest density of mutations is found in the two nucleotide bindingfields. The mutation (ΔF508) is found in 70% of cases, and 50% ofpatients are homozygous for this mutation. Seven other significantmutations are present with incidences of greater than 1%. The mutationG551D corresponds to replacement of a glycine residue (G) in position551 of the protein by an aspartic acid (D). Patients who carry thismutant have a severe pathology with a pancreatic insufficiency andserious pulmonary disorders (Cutting et al., 1990). The incidence ofobservation of this mutation reaches 3 to 5% in certain CF populations.In contrast to the ΔF508 deletion, the protein CFTR carrying themutation G551D is mature and is incorporated into the membrane (Gregoret al., 1991). However, the mutation causes an impermeability of themembrane and stimulation of the cAMP route does not open the channelassociated with expression of this mutant (Gregory, et al., 1991. Becqet al., 1994).

Other mutations, such as R117H, R334W and R347P, appear with lowincidences of 0.8, 0.4 and 0.5% respectively, and are associated with aless serious pathology (Sheppard et al., 1993). Expression of thesethree mutants generates a mature glycosylated form of the protein inharmony with its insertion into the membrane.

However, the three mutants are capable of responding to stimulation ofthe AMP route by opening of the channels. The amplitude of the currents,the unit conductance and the probability of opening of the channelassociated with each of the three mutants are modified with respect tothe normal CFTR channel (Sheppard et al., 1993. Becq et al., 1994).However, regulation by kinases/phosphatases seems normal for thesevarious mutants, including the mutants G551D and ΔF508 (Becq et al.,1994).

These observations thus show that it is possible pharmacologically toactivate a large number of CFTR mutants, including G551D and ΔF508. Inspite a lack of directing of the protein ΔF508 in the membranes ofepithelial cells affected by cystic fibrosis, several teams have shownthat this protein could be present in a functional manner in themembranes (Dalemans et al., 1991, Drumm et al., 1991. Becq et al.,1994). It therefore seems necessary and of primary importance to developa strategy for opening CFTR channels to optimize the chances of successof a treatment, but also to replace gene treatment EN,here this is notnecessary (mutations other than ΔF508).

In spite of the progress made in the genetics of cvstic fibrosis and thebiology and biochemistry of the protein CFTR, the pharmacology ofopeners of the CFTR channel is not very ell developed. Three families ofmolecules are currently put forward for their properties as activatorsor openers of the CFTR channel: phenylimidazothiazoles (levamisole andbromotetramisole), benzimidazolones (NS004) and xanthines (IBMX,theophylline . . . ).

1) The phenylimidazothiazoles (levamisole and bromotetramisole)

It has recently been shown that levamisole and bromotetramisole, byinhibiting a membrane phosphatase, allow control of the activity and thelevel of phosphorylation of the CFTR channel (Becq et al., 1994). Thesecompounds open the CFTR channel in a dose-dependent manner (Becq et al.,1996) and act on the CFTR channel with mutations at the source of thedisease (Becq et al., 1994). The mode of action of these activators isstill uncertain. These molecules do not act by the conventional routesof cAMP or of intracellular calcium. Compounds of the bromotetramisolefamily already have a therapeutic use (Grem, 1990), and levamisole isused in certain lung treatments (Van Eygen et al., 1976, Dils. 1979).The latter properties represent a certain advantage for initiatingclinical trials. However, these molecules do not, seem able to act inall cells. Intestinal cells respond poorly and opening of the CFTR afterexpression in the Xenopus ovocyte cannot be initiated. Furthermore, in atransgenic mouse model with the mutation G551D/G551D. bromotetramisoledoes not have the activator effect expected. The effects of thesemolecules therefore seem limited.

2) The benzimidazolones (NS004)

Gribkoff et al., 1994 recently demonstrated that NS004, a compound(benzimidazolone) derived from the imidazole nucleus, like levamisole,can open the channel under certain conditions (if the CFTR has beenphosphorylated). However, benzimidazolones are also activators ofnumerous potassium channels (Olesen et al., 1994) and as a result arenot very specific for CFTR.

3) The xanthines (IBMX, theophylline . . . ).

The xanthines, such as IBMX (3-isobutyl-1-methylxanthine) are CFTRactivators. The action mechanism is still poorly known and severalpossibilities exist. By inhibiting intracellular phosphodiesterases(degradation enzymes of cAMP), xanthines can increase the level of cAMPand therefore activate CFTR. Other possibilities are currently being putforward, such as binding of xanthines on the nucleotide binding fields(NBF) of CFTR.

The object of the present invention is to provide CFTR channel activatorcompounds which are more specific for CFTR than the CFTR channelactivator compounds described to date.

In this respect, the object of the present invention is to provide newmedicaments for treatment of pathologies associated with disorders intransmembrane ion flow, in particular of chlorine, in the epithelialcells of a human or animal organism.

The object of the present invention is more particularly to provide newmedicaments which can be used in the context of treatment of cvsticfibrosis, of prevention of rejection of cytotoxic drugs (in particularantitumoral drugs), or of prevention or treatment of obstructions ofbronchial routes or of digestive tracts (in particular pancreatic orintestinal), or also in the context of treatment of cardiovasculardiseases.

Another object of the present invention is to provide a preparationprocess for the compounds and pharmaceutical compositions of theinvention.

A subject of the present invention is the use of compounds of generalformula (I) Which follows:

in which:

heterocycle A is aromatic or non-aromatic, it being understood that inthe latter case the nitrogen atom of this heterocycle is linked by adouble bond to the carbon in position 4a.

R₁, R₂, R₃, R₄, R5, R₇, R8, R₉ and R₁₀, represent, independently of eachother:

a hydrogen, or bromine or fluorine atom, or

a halogen atom, in particular a chlorine atom, or

an alkyl, alkoxy, carbonyl or oxycarbonyl group, linear or branched,with approximately 1 to approximately 10 carbon atoms, these groupsbeing substituted if appropriate, in particular by a halogen, and/or bya hydroxyl, and/or by an amine (primary, secondary or tertiary), and/orby an aromatic and/or aliphatic cycle, with approximately 5 toapproximately 10 carbon atoms in the cycle, these cycles beingthemselves, if appropriate, substituted in particular by a halogen,and/or by a hydroxyl, and/or by an amine (primary, secondary ortertiary), and/or by an alkyl. alkoxy, carbonyl or oxycarbonyl group,these groups being as defined above, or

an aromatic or aliphatic cycle, with approximately 5 to approximately 10carbon atoms in the cycle, these cycles being itself, if appropriate,substituted in particular by a halogen, and/or by a hydroxyl, and/or byan amine (primary, secondary or tertiary), and/or by an alkyl, alkoxy,carbonyl or oxycarbonyl group, these groups being as defined above, or

an OR_(a) group, R_(a) representing a hydrogen atom or an alkyl,carbonyl or oxycarbonyl group, linear or branched, these groups being asdefined above, or an aromatic or aliphatic cycle, these cycles being asdefined above, or

an NR_(b)R_(c) group, R_(b) and R_(c) independently of each otherrepresenting an alkyl, alkoxy, carbonyl or oxycarbonyl group, linear orbranched, these groups being as defined above, or an aromatic oraliphatic cycle, these cycles being as defined above, or

when R₁ and R₂, or R₃ and R₄, and/or R₄ and R₅, and/or R₇ and R₈, and/orR₈ and R₉, and/or R₉ and R₁₀, do not represent the different atoms orgroups or cycles mentioned above, then R₁ in combination with R₂, or R₂in combination with R₃, and/or R₃ in combination with R₄, and/or R₄ incombination with R₅, and/or R₇ in combination with R₈, and/or R₈ incombination with R₉, and/or R₉ in combination with R₁₀, formrespectively with C₁ and C₂, or with C₂ and C₃, or with C₃ and C₄ orwith C₄, C_(4a) and C₅ or with C₇ and C₈ or with C₈ and C₉ or with C₉and C₁₀, an aromatic or aliphatic cycle, with 5 to 10 carbon atoms, ifappropriate this cycle being substituted. in particular by a halogen,and/or by an alkyl, alkoxy, carbonyl or oxycarbonyl group and/or anaromatic or aliphatic cycle, these groups or cycles being as definedabove, or

when R₃ and R₄ do not represent the different atoms or groups or cyclesmentioned above, then R₃ in combination with R₄ form an indole group offormula

in which R_(a) is as defined above,

Y represents:

an OR₄ group. R_(d) representing a hydrogen atom or an alkyl, carbonylor oxycarbonyl group, linear or branched, these groups being as definedabove, or an aromatic or aliphatic cycle, these cycles being as definedabove, or

an NR_(e)R_(i) group, R_(e) and R_(f) independently of each other,representing an alkyl, alkoxy, carbonyl or oxycarbonyl group, linear orbranched, these groups being as defined above, or an aromatic oraliphatic cycle, these cycles being as defined above,

it being understood that when R_(d), or at least one of R_(e) and R_(f)do not represent one of the different atoms or groups or cyclesmentioned above, then R_(d), or at least one of R_(e) and R_(f), incombination with R₅, or in combination with R₇, form respectively withC₅ or C₆, or with C₆, C_(6a) and C₇, an aromatic or aliphaticheterocycle with 5 to 10 carbon atoms, if appropriate substituted, inparticular by a halogen, and/or an alkyl, alkoxy, carbonyl oroxycarbonyl group, and/or an aromatic or aliphatic cycle, these groupsor cycles being as defined above,

n is equal to 0 or 1, with:

when n is equal to 0:

 X represents an atom in anionic form, such as a halogen atom, inparticular a bromine or chlorine atom, or a group of atoms in anionicform, such as a perchlorate, and the nitrogen of heterocycle A offormula (I) is in quaternary form and is linked on the one hand bycovalent bond to the carbon in position 11, and, on the other hand, byionic bond to X defined above,

 it being understood that when R₁ and R₁₀ do not represent the differentatoms or groups or cycles mentioned above, then R₁ in combination withR₁₀ form with C₁, the nitrogen of heterocycle A of formula (I), C₁₁, andC₁₀, an aromatic or aliphatic heterocycle with 5 to 10 carbon atoms, ifappropriate substituted, by a halogen, and/or an alkyl, carbonyl oroxycarbonyl group, and/or by an aromatic or aliphatic cycle, thesegroups or cycles being as defined above,

when n is equal to 1, then X represents a hydrogen atom, or a halogenatom, in particular a bromine, or chlorine, or fluorine atom.

For the preparation of medicaments intended for the treatment ofpathologies in particular pulmonary, digestive or cardiac, linked totransmembrane ion flow disorders, in particular chlorine and, ifappropriate, bicarbonate, in the organism (human or animal), inparticular for the preparation of medicaments intended for the treatmentof mucoviscidosis, or for the prevention of rejection of cytotoxic drugs(in particular antitumoral), for the treatment of obstructions to thebronchial routes or digestive tracts (in particular pancreatic orintestinal).

A more particular subject of the invention is the use as describedabove, of compounds of general formula (I) in which n=1, andcorresponding to the derivatives of general formula (11) which follow:

in which R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, X and Y are as definedabove.

Therefore, the invention also more particularly relates to the use asdescribed above, of the compounds of general formula (IIa) whichfollows:

in which

R₁ and R₂ represent a hydrogen atom, or form in combination with C₁ andC₂ an aromatic cycle with 6 carbon atoms,

Y represents an —OH or NH₂ group,

R₇, R₈, R₉ and R₁₀ represent a hydrogen atom, or one of R₇, R₈, R₉ orR₁₀, represents a halogen atom, in particular a bromine, chlorine orfluorine atom,

X represents a hydrogen atom or a halogen atom, in particular a bromine,chlorine or fluorine atom.

The compounds of general formula (IIa) advantageously used within thescope of the present invention, are those chosen from the following:

More particularly the invention also relates to the use as describedabove, of compounds of general formula (IIb) which follows:

in which R₃, R₁, R₂, R₅, R₇, R₈, R₉, R₁₀, X and Y are as defined above,and in particular the compounds of formula (IIb) in which:

R_(a) represents a hydrogen atom,

R₁ and R₂ represent a hydrogen atom, and there is no double bond betweenthe two carbons carrying R₁ and R₂,

R₅ represents a hydrogen atom,

R₇, R₈, R₉, and R₁₀ represent a hydrogen atom, or one of R₇, R₈, R₉, andR₁₀ represents a halogen atom, in particular a chlorine, bromine orfluorine atom,

Y represents —NH₂,

X represents a halogen atom, in particular a bromine, chlorine orfluorine atom.

The compounds of formula (IIb) advantageously used within the scope ofthe present invention, are those chosen from the following:

compound A: R₇═Cl, R₈═R₉═R₁₀═H,

compound B: R₇═R₈═R₉═R₁₀═H,

compound C: R₈═Cl, R₇═R₉═R₁₀═H,

compound D: R₉═Cl, R₇═R₈═R₁₀═H,

compound E: R₁₀═Cl, R₇═R₈═R₉═H,

compound F: R₉═Br, R₇═R₈═R₁₀═H.

A more particular subject of the invention is the use as describedabove, for the compounds of general formula (I) in which n=0, andcorresponding to benzo[c]quinolizinium derivatives of formula (III)which follows:

in which R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, X and Y are as definedabove.

Therefore, the invention more particularly relates to the use asdescribed above, of the compounds of general formula (IIIa) whichfollows:

in which:

R₁ and R₂ represent a hydrogen atom, or form in combination with C₁ andC₂ an aromatic cycle with 6 carbon atoms,

Y represents an —OH or —NH₂ or NHCOCH₃ group,

R₇, R₈, R₉ and R₁₀, represent a hydrogen atom or one of R₇, R₈, R₉ andR₁₀ represents a halogen atom, in particular a chlorine, bromine orfluorine atom,

X represents a halogen atom in anionic form, in particular a bromineatom Br⁻, or a chlorine atom Cl⁻, or a group of atoms in anionic form,in particular a perchlorate ClO₄—.

Particularly preferred compounds within the scope of the presentinvention are those of formula (IIIa) in which:

R₁ and R₂ represent a hydrogen atom,

Y represents an —OH group,

X represents a halogen atom in anionic form, in particular a bromineatom Br⁻, or a chlorine atom Cl⁻, or a group of atoms in anionic form,in particular a perchlorate ClO₄ ⁻,

R₇, R₈, R₉ and R₁₀, represent independently of each other a hydrogenatom or a halogen atom, in particular a chlorine, bromine or fluorineatom.

Compounds of general formula (IIIa) advantageously used within the scopeof the present invention are those chosen from the following:

More particularly the invention relates to the use as described above,of compounds of general formula (IIIb) which follows:

in which R₃, R₁, R₂, R₅, R₇, R₈, R₉, R₁₀, X and Y are as defined above,and in particular the compounds of formula (IIIb) in which:

R_(a) represents a hydrogen atom,

R₁ and R₂ represent a hydrogen atom, and there is no double bond betweenthe two carbons carrying R₁ and R₂,

R₅ represents a hydrogen atom,

R₇, R₈, R₉, and R₁₀ represent a hydrogen atom, or one of R₇, R₈, R₉, andR₁₀ represents a halogen atom, in particular a chlorine, bromine orfluorine atom.

Y represents —NH₂,

X represents a halogen atom in anionic form, in particular a bromineatom Br⁻, or a chlorine atom Cl⁻, or a group of atoms in anionic form,in particular a perchlorate ClO₄ ⁻,

The compounds of formula (IIIb) advantageously used within the scope ofthe present invention, are those chosen from the following:

compound G: R₇═Cl, R₈═R₉═R₁₀═H⁻,

compound H: R₇═R₈═R₉═R₁₀═H,

compound I: R₈═Cl, R₇═R₉═R₁₀═H,

compound J: R₉═Cl, R₇═R₈═R₁₀═H,

compound K: R₁═Cl, R₇═R₈═R₉═H,

compound L: R₉═Br, R₇═R₈═R₁₀═H.

A subject of the invention is any pharmaceutical composition containing,as active ingredient(s), at least one of the compounds of generalformula (I) described above, in combination with a physiologicallyacceptable vehicle.

A more particular subject of the invention is an%, pharmaceuticalcomposition as described above containing, as active ingredient(s), atleast one of the compounds of formula (II) and more particularly offormula (IIa), as described above, and in particular at least one ofcompounds 1 to 10 described above, and even more particularly of formula(IIb), as defined above, and in particular at least one of compounds Ato F described above.

A more particular subject of the invention is any pharmaceuticalcomposition as described above containing, as active ingredient(s), atleast one of the compounds of general formula (III) and moreparticularly of formula (IIIa), described above, and in particular atleast one of compounds 11 to 27 described above, and even moreparticularly of formula (IIIb), as defined above, and in particular atleast one of compounds G to L described above.

Preferred pharmaceutical compositions of the invention are thosecontaining compound 19 (also designated MPB-07), if appropriate incombination with one (or more) other compound(s) of the inventiondescribed above.

Advantageously, the pharmaceutical compositions according to theinvention are presented in a form which can be administered by oralroute, in particular in the form of tablets or capsules, or in a formwhich can be administered by parenteral route, in particular in the formof preparations which are injectable by intravenous, intramuscular orsub-cutaneous route, or also via the airways, in particular by pulmonaryroute in the form of aerosols.

Yet more advantageously, the pharmaceutical compositions according tothe invention are characterized in that the quantities of activeingredient(s) are such that the daily dose of active ingredient(s) isapproximately 0.1 mg/kg to 5 mg/kg, in particular approximately 3 mg/kg,in one or more doses.

The invention also relates to the compounds of general formula (I)described above, as such, with the exception of compounds 2, 3, 9, 10,11 (or MPB-26), 12 or (MPB-5), 22, 23 and 24 described above.

More particularly a subject of the invention is the compounds of generalformula (I) described above, in which n=1, and corresponding to thecompounds of general formula (II) described above, with the exception ofcompounds 2,3,9 and 10.

More particularly the invention relates to the compounds of generalformula (la) described above, of which in particular compounds 1, 4, 5,6, 7 and 8 described above.

More particularly the invention also relates to the compounds of generalformula (IIa) described above, of which in particular compounds A to Fdescribed above.

More particularly a subject of the invention is the compounds of generalformula (I) described above, in which n=0, and corresponding to thebenzo[c]quinolizinium derivatives of formula (III) described above, withthe exception of compounds 11, 12, 22, 23 and 24.

More particularly the invention relates to the compounds of generalformula (IIIa) described above, of which in particular compounds 13, 14,15, 16, 17, 18, 19, 20, 21, 25, 26 and 27 described above.

More particularly the invention also relates to the compounds of generalformula (IIIb) described above, of which in particular compounds G to Ldescribed above.

A subject of the invention is also a preparation process for thecompounds of general formula (I), characterized in that it includes thefollowing steps:

treatment of the derivative of formula (A) in which R₁, R₂, R₃, R₄ andR₅ are as described in formula (I), with phenyllithium or lithiumdiisopropyl amide, advantageously in ether or THF, which leads to theobtaining of derivatives of formula (B) in which R₁, R₂, R₃, R₄ and R₅are as described in formula (I), according to the following reactiondiagram:

condensation of the derivative of formula (B) obtained in the previousstage with the derivative of formula (C) in which R₇, R₈, R₉, R₁₀ and Xare as defined in formula (I), which leads to the obtaining ofderivatives of formula (B) in which R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀and X are as defined in formula (I), according to the following reactiondiagram:

treatment of the compound of formula (D) by the addition of H₂O, whichleads to the obtaining of the following derivative of formula (II),corresponding to a derivative of formula (II) described above, in whichR₁ to R₅, R₇ to R₁₀, and X are as defined in formula (I), and Yrepresents —NH₂,

if appropriate, treatment of the above-mentioned compound of formula(II), with a derivative containing the R_(e) and R_(f) groups as definedin formula (I), this derivative being capable of reacting with thenitrogen atom linked to the carbon in position 6 of the above-mentionedcompound of formula (II), in particular by a halide of R_(e) and/orR_(f) while having, if necessary, taken care to protect beforehand thoseother functions present on the above-mentioned compound of formula (II)and capable of reacting with the derivative containing theabove-mentioned R_(e) and R_(f) groups, which leads to the obtaining ofthe following compound of formula (II) in which R₁ to R₅, R₇ to R₁₀ andX are as defined above and Y represents an —NR_(e)R_(f) group as definedin formula (I),

if appropriate, hydrolysis, in particular by the action of sulphuricacid (pH3) at 40° C., of the above-mentioned compound of formula (II) inwhich Y represents NH₂, which leads to the obtaining of the followingcompound of formula (II), corresponding to a derivative of formula (II)described above, in which R₁ to R₅, R₇ to R₁₀ and X are as defined informula (I), and Y represents an —OH group,

if appropriate, treatment of the above-mentioned compound of formula(II), in which Y represents an —OH group, with a derivative containingthe R_(d) group, as defined in formula (I), this derivative beingcapable of reacting with the oxygen atom 71 linked to the carbon inposition 7 of the above-mentioned compound of formula (II), inparticular by a halide of R_(d), while having, if necessary, taken careto protect beforehand those other functions present on theabove-mentioned compound of formula (II) and capable of reacting withthe derivative containing the above-mentioned R_(d) group, which leadsto the obtaining of the following compound of formula (II) in which R₁to R₅, R₇ to R₁₀ and X are as defined above and Y represents an —OR_(d)group as defined in formula (I),

if appropriate, heating, advantageously at 200° C., the above-mentionedcompounds of formula (II) in which Y represents —NH₂ or —OH, which leadsrespectively to the following compounds of formula (III) describedabove, corresponding to the compounds of formula (III) described above,in which R₁ to R₅, R₇ to R₁₀ and X are as defined in formula (I) and Yrepresents an —NH, or —OH group,

if appropriate, treatment of the above-mentioned compound of formula(III), in which Y represents an —NH₂ group, with a derivative containingthe R_(e) and R_(f) groups as defined in formula (I), this derivativebeing capable of reacting with the nitrogen atom linked to the carbon inposition 6 of the above-mentioned compound of formula (III), inparticular by a halide of R_(e) and/or R_(f) while having, if necessary,taken care to protect beforehand those other functions present on theabove-mentioned compound of formula (III) and capable of reacting withthe derivative containing the above-mentioned R_(e) and R_(f) groups,which leads to the obtaining of the following compound of formula (III)in which R₁ to R₅, R₇ to R₁₀ an X are as defined above and Y representsan —NR_(e)R_(f) group as defined in formula (I),

if appropriate, treatment of the above-mentioned compound of formula(III), in which Y represents an —OH group, with a derivative containingthe R_(d) group, as defined in formula (I), this derivative beingcapable of reacting with the oxygen atom linked to the carbon inposition 7 of the above-mentioned compound of formula (III), inparticular by a halide of R_(d), while having, if necessary, taken careto protect beforehand those other functions present on theabove-mentioned compound of formula (III) and capable of reacting withthe derivative containing the above-mentioned R_(d) group, which leadsto the obtaining of the following compound of formula (III) in which R₁to R₅, R₇ to R₁₀ and X are as defined above and Y represents an —OR₄group as defined in formula (I),

if appropriate, hydrogenation of the above-mentioned compounds offormulae (II) or (III), in particular by catalytic hydrogenation in thepresence of platinum oxide at reduced pressure, which leads to theobtaining of the following compounds of formulae (II) or (III):

in which R₁ to R₅, R₇ to R₁₀, X and Y are as defined above.

Compounds A to L described above are advantageously obtained by thetreatment of harmalane with butyl lithium (BuLi, 2 eq.) at −40° C., thenthe addition of 2-chlorobenzonitrile (if appropriate substituted by oneor more R₇, R₈, R₉ and R₁₀ groups as defined above), which leads to theobtaining of the compounds of formula A to F which, by heating at 195°C., under nitrogen, leads respectively to compounds G to L.

The invention will be further illustrated using the detailed descriptionwhich follows of preparation processes for compounds 1 to 24 describedabove, as well as the study of the effects of certain of these compoundson CFTR.

I—Preparation Processes for Compounds 1 to 24

a) 1-Hydroxy, 1-Phenyl, 2-(2-Pyridyl)ethylene (Compound 1)

Compound 1 is prepared according to any operating method identical tothat described hereafter within the scope of the preparation of compound2, but using benzonitrile instead of 2-chlorobenzonitrile.

b) I-Hydroxy, 1-(2-Chlorophenyl, 2-(2-Pyridyl)ethylene (Compound 2)

2.22 g (0.022 mole) of diisopropylamine in 30 ml of anhydrous THF isplaced in a 500 ml reactor, equipped with, a reflux condenser with acalcium chloride drying tube and a supply of nitrogen. The solution istaken to 0° C., then 13.75 ml of BuLi in solution at 1.6M of hexane(0.022 mole) is added. Agitation is carried out for 30 minutes at 0° C.,then the temperature is lowered to −40° C., then 1.86 g (0.02 mole) of2-methylpyridrine is added. Agitation is carried out for 30 minutes at−40° C., then 2.75 g of 2-chlorobenzonitrile in 20 ml of anhydrous THFis added. The reaction medium is left to return to ambient temperatureand 20 ml of water is added, then the pH is adjusted to 2 be theaddition of 2N H₂SO₄. Heating under reflux is carried out underagitation for 1 hour, followed by extraction with chloroform and dryingover sodium sulphate. The solvent is evaporated off and the residuedissolved in the minimum amount of anhydrous ether, then ethanolsaturated with HCl is added dropwise until the complete precipitation ofthe hydrochloride. In this way 2.99 g of compound 2 is obtained i.e, ayield of 56%.

Melting point (M.p.) = 190° C. Elementary analysis: C₁₃H₁₁ClN₂OCalculated % C: 58.20 H: 4.10 N: 5.20 Found % C: 58.00 H: 4.10 N: 5.30

c) 1-Amino, 1-(2-Chlorophenyl, 2-(2-Pyridyl)ethylene (Compound 3)

Compound 3 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 2,but without the hydrolysis stage by the addition of H₂SO₄.

d) 1-Hydroxy, 1-(2-Bromophenyl, 2-(2-Pyridyl)ethylene (Compound 4)

Compound 4 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 2,but using 2-benzobenzonitrile instead of 2-chlorobenzonitrile.

e) 1-Hydroxy, 1-(2,3-Dichlorophenyl, 2-(2-Pyridyl)ethylene (Compound 5)

2.22 g (0.022 mole) of diusopropylamine in 30 ml of anhydrous THF isplaced in a 500 ml reactor, equipped with a reflux condenser with acalcium chloride drying tube and a supply of nitrogen. The solution istaken to 0° C., then 13.75 ml of BuLi in solution at 1.6M of hexane(0.022 mole) is added. Agitation is carried out for 30 minutes at 0° C.,then the temperature is lowered to −40° C., then 1.86 g (0.02 mole) of2-methylpyridrine is added. Agitation is carried out for 30 minutes at−40° C., then 2.58 g (0.015 mole) of 2,3-dichlorobenzonitrile in 20 mlof anhydrous THF is added. The reaction medium is left to return toambient temperature and 20 ml of water is added, then the pH is adjustedto 2 by the addition of 2N H₂SO₄. Heating under reflux is carried outunder agitation for 2 hours, followed by separation of the organic phaseand drying over sodium sulphate. The solvent is evaporated off and theresidue is chromatographed on a silica column eluting with chloroform inorder to obtain 3.19 g (80%) of pure ketone.

Melting point (M.p.) = 112° C. Elementary analysis: C₁₃H₉NOCl₂Calculated % C: 58.67 H: 3.41 N: 5.26 Found % C: 58.53 H: 3.63 N: 5.34

¹H NMR spectrum (CDCl₃) (δ ppm, signal, N protons, attribution): 8,doublet, H in position 6 of pyridine: 7,6,5, multiplet, 6, aromatic H's.5.55, s, b (80%) vinylic H: 4.25, s s2 (20%) CH₂.

The base obtained in this way can be converted into the hydrochloride:the base is dissolved in anhydrous ether and anhydrous ethanol saturatedwith HCl is added dropwise.

Elemental analysis: C_H₁₀Cl₃ Calculated % C: 51.60 H: 3.33 N: 4.63 Found% C: 51.75 H: 3.52 N: 4.58

f) 1-Hydroxy, 1-(2,4-Dichlorophenyl), 2-(2-Pyridyl)ethylene (Compound 6)

Compound 6 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 2,but using 2-4-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

g) 1-Hydroxy, 1-(2,5-Dichlorophenyl), 2-(2-Pyridyl)ethylene (Compound 7)

Compound 7 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 2,but using 2-5-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

h) 1-Hydroxy, 1-(2,6-Dicholorphenyl), 2-(2-Pyridyl)ethylene (Compound 8)

Compound 8 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 2,but using 2-6-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

i) 1-Hydroxy, 1-(2-Chlorophenyl), 2-(2-Quinonyl)ethylene (Compound 9)

Compound 9 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 2,but using 2-methylquinoline instead of 2-methylpyridine.

j) 1-Amino, 1-(2-Chlorophenyl, 2-(2-Quinolyl)ethylene (Compound 10)

Compound 10 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 9,but without the hydrolysis stage by the addition of H₂SO₄.

k) 6-Aminobenzo[c]quinolizium MPB-26 (Compound 11)

2.22 g (0.022 mole) of diusopropylamine in 30 ml of anhydrous THF isplaced in a 500 ml reactor, equipped with a reflux condenser with acalcium chloride drying tube and a supply of nitrogen. The solution istaken to 0° C., then 13.75 ml of BuLi in solution at 1.6M in hexane(0.022 mole) is added. Agitation is carried out for 30 minutes at 0° C.,then the temperature is lowered to −40° C., then 1.86 g (0.02 mole) of2-methylpyridrine is added. Agitation is carried out for 30 minutes at−40° C., then 2.75 g of 2-chlorobenzonitrile (0.02 mole) in 20 ml ofanhydrous THF is added. The reaction medium is left to return to ambienttemperature and a 10% solution of ammonium chloride is added. Theorganic phase is separated, dried over Na₂SO₄, evaporated and theresidue is taken to 200°° C., under a current of nitrogen for 15minutes. The evolution of a white vapour is observed and the residuesolidifies to a brownish mass. The product is purified by a firstwashing with acetone then dissolution in ethanol and precipitation withethyl acetate. Purification can also be carried out with chromatographyon a neutral alumina column eluting with ethyl acetate. In this way 1.73g (35%) of product is obtained crystallizing with one molecule of water.

Melting point (M.p.) = decomposition at approx. 280° C. Elementaryanalysis: C₁₃ H₁₃C₁N₂O (C₁₃ H₁₁C₁N₂, H₂O) Calculated % C: 62.78 H: 5.27N: 11.26 Found % C: 62.86 H: 5.03 N: 11.25 Mass spectrum: m/e 194(M⁺—HCl—H₂O). Infrared spectrum (KBr) (ν cm⁻¹, attribution): 3460, 3360,NH₂: 1660, 1640, 1660, 1640, 1660, C═N, C═C: 760 orthosubstitutedbenzene.

¹H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution): 3.2 to4 exchangeable peak D₂O (NH₂+H₂O); 7.1 singlet, H in position 5: 7.4 to8.15, 2 massives, aromatic 5H's: 9.00, 2H, aromatics; 9.8, doublet J=8Hz, H in position 1.

l) 6-Hydroxybenzo[c]quinolizinium Chloride MPB-05 (Compound 12)

1-hydroxy, 1-(2-chlorophenyl), 2-(2-pyridyl)ethylene (compound 2) isneutralized with an aqueous solution of sodium carbonate, the base isextracted with ether, the solution is dried over Na₂SO₄ then the solventis evaporated off, 1.16 g (0.005 mole) of base in the form of a paleyellow oil is heated under nitrogen at 195° C. for 15 minutes. Theresidue thus obtained is washed with acetone, then dissolved in ethanoland precipitated by the addition of ethyl acetate, 0.75 g (60%) ofproduct is obtained crystallized with one molecule of water and with acreamy white colour.

Melting point (M.p.) = 256° C. (decomposition) Elementary analysis:C₁₃H₁₀ClNO, H₂O i.e. C₁₃H₁₂ClNO (M = 231.5 + 18 = 249.5) Calculated % C:62.53 H: 4.85 N: 5.61 Found % C: 62.40 H: 5.00 N: 5.80 Mass spectrum:m/e 195 (M⁻—HCl—H₂O), 167 (M⁺—HCl—H₂O—CO). Infrared spectrum (KBr) (νcm⁻¹, attribution): 3250, OH: 1640, C═N, 770 orthosubstituted benzene.

¹H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution):

6.60 broad exchangeable peak D₂O, HO; 7.75, singlet, H in position 5:7.9 to 18.6, multiplet, 6H aromatics: 9.15 doublet, J=6.5 Hz, 1H: 10,doublet, J=6 Hz, H in position 1.

m) 6-Aminobenzo[c]quinolizinium Bromide MPB-01 (Compound 13):

Compound 13 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 11,but using 2-bromobenzonitrile instead of 2-chlorobenzonitrile.

n) 6-Amino, 10-chlorobenzo[c]quinolizinium Chloride MPB-02 (Compound14):

Compound 14 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 11,but using 2,3-dichlorobenzonitrile instead of 2-chlorobenzonitrile,

o) 6-Amino, 9-Chlorobenzo[c]quinolizinium Chloride (Compound 15):

Compound 15 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 11,but using 2,4-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

p) 6-Amino, 7-Chlorobenzo[c]quinolizinium Chloride (Compound 16):

Compound 16 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 11,but using 2.6-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

q) 6-Amino, 8-Chlorobenzo[c]quinolizinium Chloride (Compound 17):

Compound 17 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 11,but using 2,5-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

r) 6-Hydroxbenzo[c]quinolizinium Bromide MPB-06 (Compound 18):

Compound 18 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 19,but is carried out starting from compound 4 instead of compound 5.

s) 6-Hydroxy, 10-Chlorobenzo[c]quinolizinium Chloride MPB-07 (Compound19):

1.40 g (0.0053 mole) of 1-hydroxy, 1-(2-bromophenyl),2-(2-pyridyl)ethylene (compound 5) is heated under nitrogen at 215° C.At around 190°° C. the appearance of white fumes of HCl is noted andheating is continued at 220°° C. for 10 minutes. The product is washedwith chloroform then the residue (1.82 g) is purified by chromatographyon a silica column eluting with acetate and alcohol. In this way 0.58 g(42%) of product is obtained.

Melting point (M.p.) = 196° C. (decomposition) Elementary analysis:C₁₃H₁₀NOCl₂ Calculated % C: 56.75 H: 3.66 N: 4.63 Found % C: 56.25 H:3.31 N: 4.78

t) 6-Hydroxy, 9-Chlorobenzo[c]quinolizinium Chloride MPB-08 (Compound20):

Compound 20 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 19,but is carried out starting from compound 6 instead of compound 5.

u) 6-Hydroxy, 7-Chlorobenzo[c]quinolizinium Chloride (Compound 21):

Compound 21 is prepared according to any operating method identical tothat described above within the scope of the preparation of compound 19,but is carried out starting from compound 8 instead of compound 5.

v) 8-Aminodibenzo[c.f]quinolizinium Perchlorate (Compound 22):

2.22 g (0.022 mole) of diusopropylamine in 30 ml of anhydrous THF isplaced in a 500 ml reactor, equipped with a reflux condenser with acalcium chloride drying tube and a supply of nitrogen. The solution istaken to 0° C., then 13.75 ml of BuLi in solution at 1.6M in hexane(0.022 mole) is added. Agitation is carried out for 30 minutes at 0° C.then the temperature is lowered to −40° C. then 2.86 g (0.02 mole) ofquinaldine is added. Agitation is carried out for 30 minutes at −40° C.,then 2.75 g of 2-chlorobenzonitrile (0.02 mole) in 20 ml of anhydrousTHF is added. The reaction medium is left to return to ambienttemperature and a 10% solution of ammonium chloride is added. Theorganic phase is separated, dried over Na₂SO₄, evaporated and theresidue is taken to 230° C. under a current of nitrogen for 30 minutes.The evolution of a white hydrochloric acid vapour is observed and theresidue solidifies to a brownish mass. The product is purified by afirst washing with acetone then dissolution in ethanol and precipitationwith ethyl acetate. The product is dissolved in a minimum amount ofwater and a solution of perchloric acid is added until precipitation iscomplete. The compound is filtered out in order to recover 2.20 g (32%).

Elementary analysis: C₁₇H₁₃ClN₂O₄ Calculated % C: 59.22 H: 3.80 N: 8.13Found % C: 59.39 H: 3.95 N: 8.26 Infrared spectrum (KBr) (ν cm⁻¹,attribution): 3400, 3280, NH₂: 1650, 1600, 1000, broad band, ClO₄

¹H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution): 7.0,singlet, 1H in position 5; 7.5 to 8.7, multiplet, aromatic 10H's: 9.0singlet, 2H, exchangeable D₂O.

w) 6-Acetoamidobenzo[c]quinolizinium Chloride (Compound 23)

1 g (0.004 mole) of 6-aminobenzo[c]quinolizinium is dissolved in 10 mlof acetic acid, then 25 ml of acetic anhydride is added. The reactionmedium is taken to reflux for 24 hours, then the acetic anhydride andacetic acid is evaporated off under reduced pressure.

The product obtained has a violet colour and is washed with ethylacetate, then recrystallized from ethanol, 0.93 g (85%) of a creamywhite product is obtained.

M.p.=>280° C.;

Elementary analysis: C₁₅H₁₃ClN₂O Calculated % C: 66.05 H: 4.80 N: 10.27Found % C: 65.85 H: 5.01 N: 10.3 Infrared spectrum (KBr) (ν cm⁻¹,attribution): 3450, NH: 1700, C═O.

x) 1,2,3,4-Tetrahydro, 6-Aminobenzo[c,f]quinolizinium Perchlorate(Compound 24):

0.50 g (0.002 mole) 6-aminobenzo[c]quinolizinium hydrochloride isdissolved in 20 ml of ethanol and is placed in the presence of platinumoxide and hydrogen at atmospheric pressure. Hydrogenation is carried outover a few minutes then the solution is filtered, the alcohol isevaporated off and the residue is taken up in 10 ml of distilled waterand a 25% solution of perchloric acid is added while the precipitateforms. The white product is recrystallized from methanol in order toproduce 0.54 g (91%) of perchlorate.

M.p. = 240° C. Elementary analysis: C₁₃H₁₅ClN₂O₄ Calculated % C: 52.27H: 5.06 N: 9.38 Found % C: 52.30 H: 5.16 N: 9.46 Infrared spectrum (KBr)(ν cm⁻¹, attribution): 3460, 3360, NH₂: 1650, 1600, 1100, broad band,CLO₄.

¹H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution):

2.1, multiplet. CH₂ in position 2 and 3; 3.20, triplet, CH, in position4: 4.45, triplet, CH₂ in position 1; 6.60, singlet. H in position 5; 7.6to 8.4, multiplet, 4H, aromatics; 8.6 singlet, 2H, exchangeable D₂O.

II-Preparation Process for Compounds A to F

a) 1-Amino 1-(2,6-Dichlorophenyl)2-[1-(3,4-Dihydropyrido[3-4-b]indolyl)]ethylene (Compound A)

1.84 g (0.01 mole) of harmalane is solubilized in 40 ml of THF in areactor equipped with a nitrogen supply and the reaction medium is takento −40° C., 13.75 ml (0.022 mole) of BuLi is added dropwise, a dark redcolouring appears, the solution is left under agitation for 30 minutes,1.71 g (0.01 mole) of 2,6-dichlorobenzonitrile is solubilized in 15 mlof THF, then added dropwise. After 1 hour at −40° C., the mixture isagitated for 4 hours at room temperature. The development of thereaction is monitored by TLC, the disappearance of the starting productsis correlated to the appearance of a yellow fluorescent spotcharacteristic of the imine. Hydrolysis with 5 ml of a 10% solution ofNH₄Cl allows the THF phase containing the imine to be recovered. Thisphase is dried over Na₂SO₄, filtered then evaporated to dryness. Theproduct is purified by column chromatography on silica gel, eluting witha CH₂Cl₂/CH₃COOC₂H₅ 5% mixture. In this way 2.06 g of compound A isobtained, i.e, a yield of 58%.

M.p. = 228° C. Elementary analysis: C₁₉H₁₅N₃Cl₂ Calculated % C: 64.06 H:4.24 N: 11.79 Found % C: 64.21 H: 4.37 N: 11.62

Infrared spectrum (KBr) (ν cm⁻¹, attribution): 3428, 3255, 3134 cm⁻¹.NH: NH₂; 3134 cm⁻¹, C═C—H; 2932, 2843 cm⁻¹, CH₂; ¹H NMR spectrum(DMSOd6) (δ ppm, signal, n protons, attribution): 8.40 singletexchangeable by D₂O, 3 H, NH and NH₂; 7.20, multiplet, 7 H, aromaticprotons; 5.00, singlet, 1H, vinylic H; 3.70, triplet, J=6 Hz, 2 H, CH₂;3.00, triplet, J=6 Hz, 2 H, CH₂.

b) 1-Amino 1-(O-chlorophenyl)2-[1-(3,4-Dihydropyrido[3-4-b]indolyl)]ethylene (Compound B)

Compound B is prepared according to any operating method identical tothat described above within the scope of the preparation of compound A,but using orthochlorobenzonitrile instead of 2,6-dichlorobenzonitrile.

c) 1-Amino 1-(2,5-Dichlorophenyl)2-[1-(3,4-Dihydropyrido[3-4-b]indolyl)]ethylene (Compound C)

Compound C is prepared according to any operating method identical tothat described above within the scope of the preparation of compound A,but using 2,5-dichlorobenzonitrile instead of 2,6-dichlorobenzonitrile.

d) 1-Amino 1-(2,4-Dichlorophenyl)2-[1-(3,4-Dihydropyrido[3-4-b]indolyl)]ethylene (Compound D)

Compound D is prepared according to any operating method identical tothat described above within the scope of the preparation of compound A,but using 2,4-dichlorobenzonitrile instead of 2,6-dichlorobenzonitrile.

e) 1-Amino 1-(2,3-Dichlorophenyl)2-[1-(3,4-Dihydropyrido[3-4-b]indolyl)]ethylene (Compound E)

Compound E is prepared according to any operating method identical tothat described above within the scope of the preparation of compound A,but using 2,3-dichlorobenzonitrile instead of 2,6-dichlorobenzonitrile.

f) 1-Amino 1-(4-Bromo, 2-Chlorophenyl)2-[1-(3,4-Dihydropyrido[3-4-b]indolyl)]ethylene (Compound F)

Compound F is prepared according to any operating method identical tothat described above within the scope of the preparation of compound A,but using 2-chloro-4 bromobenzonitrile instead of2,6-dichlorobenzonitrile.

III—Production Process for Compounds G to L

Cyclization of compounds A to F into quinolizinium G to L.

a) 14-Amino 6,7-Dihydro 12H-1-Chloro Benzo-[n]indolo[2,3-a]quinoliziniumChloride (Compound G).

Purified enamine A is heated under nitrogen; this product liquefies atabout 150° C., then at 195°° C. white fumes appear and the productsolidifies. Heating is maintained at this temperature for 10 minutes.The following is observed using TLC: disappearance of the yellowfluorescent spot of the imine with an Rf of 0.3 on silica in CH₂Cl₂ andthe appearance of a yellow-green fluorescent spot of the cyclizedproduct with an Rf of 0.1 on alumina in alcohol. The product is purifiedby washing with acetone, then recrystallization from alcohol or bychromatography: alumina—alcohol.

The product thus obtained has a light brown colour, with a yield of 17%.

M.p. = 228° C. Elementary analysis: C₁₉H₁₅N₃Cl₂, ½ H₂O Calculated % C:62.48 H: 4.41 N: 11.50 Found % C: 62.29 H: 4.47 N: 11.43

¹H NMR spectrum (CF₃COOD) (δ ppm, signal, n protons, attribution):8.10-6.20, massive, 11 H, aromatic protons+NH+NH₂; 3.90, triplet poorlyresolved, 2 H, CH₂ in position 6; 3.00, triplet poorly resolved, 2 H,CH₂ in position 7; Infrared spectrum (KBr) (ν cm⁻¹, attribution)presenting all the same absorptions: 3458, 3371 cm⁻¹, NH: NH₂; 3073cm⁻¹; C═C—H; 1638 cm⁻¹, C═N, C═C.

b) 14-Amino 6,7-Dihydro 12H-Benzo-[f]indolo[2,3-a]quinolizinium Chloride(Compound H).

Compound H is prepared according to any operating method identical tothat described above within the scope of the preparation of compound G,but using compound B instead of compound A.

The product obtained has a mustard yellow colour with a yield of 56%.

M.p. = greater than 260° C. Elementary analysis: C₁₉H₁₆N₃Cl Calculated %C: 70.91 H: 5.01 N: 13.06 Found % C: 70.33 H: 5.06 N: 12.71

c) 14-Amino 6,7-Dihydro 12H-2-Chloro Benzo-[1]indolo[2.3-a]quinoliziniumChloride (Compound I).

Compound I is prepared according to any operating method identical tothat described above within the scope of the preparation of compound G,but using compound C instead of compound A.

The product obtained has a light brown colour with a yield of 7%.

M.p. = greater than 260° C. Elementary analysis: C₁₉H₁₅N₃Cl₂, H₂OCalculated % C: 60.97 H: 4.38 N: 11.22 Found % C: 61.32 H: 5.03 N: 10.52

d) 14-Amino 6,7-Dihydro 12H-3-Chloro Benzo-[1]indolo[2,3-a]quinoliziniumChloride (Compound J).

Compound J is prepared according to any operating method identical tothat described above within the scope of the preparation of compound G,but using compound D instead of compound A.

The product obtained has a light brown colour with a yield of 52%.

M.p. = greater than 260° C. Elementary analysis: C₁₉H₁₅N₃Cl₂ Calculated% C: 64.06 H: 4.24 N: 11.79 Found % C: 63.89 H: 4.48 N: 11.58

e) 14-Amino 6,7-Dihydro 12H-4-Chloro Benzo-[1]indolo[2,3-a]quinoliziniumChloride (Compound K).

Compound K is prepared according to any operating method identical tothat described above within the scope of the preparation of compound G,but using compound E instead of compound A.

The product obtained has a light brown colour with a yield of 6%.

M.p. greater than 260° C. Elementary analysis: C₁₉H₁₅N₃Cl₂, H₂OCalculated % C: 60.97 H: 4.58 N: 11.22 Found % C: 60.56 H: 4.66 N: 10.84

f) 14-Amino 6,7-Dihydro 12H-3-Bromo Benzo-[1]indolo[2,3-a]quinoliziniumChloride (Compound L).

Compound L is prepared according to any operating method identical tothat described above within the scope of the preparation of compound G,but using compound F instead of compound A.

The product obtained has a mustard yellow colour with a yield of 12%.

M.p. = greater than 260° C. Elementary analysis: C₁₉H₁₅N₃Cl, Br, 2H₂OCalculated % C: 52.25 H: 4.38 N: 9.62 Found % C: 52.28 H: 4.36 N: 9.66

IV—Studs of the Effects of Compounds of the Invention on CFTR

A) Methodology

a) Culture of Human Epithelial Cells and Recombinant CHO Cells:

Several types of cells are used for this study: the intestinal linesT84, Caco-2 and HT 29. Chinese hamster ovary (CHO-K1) cells weretransfected with the aid of the vector pNUT (Tabcharani et al., 1991)with or without (control cells) incorporation of normal or mutated CFTRcDNA (Chang et al., 1993). The cells are kept in this specific mediumand then inoculated at a low density on to glass slides and cultured at37° C. (5% CO₂), before the patch clamping experiments. The survivalmedium of the cells is composed of αMEM with foetal calf serum (7%) andantibiotics: 50 IU/ml penicillin and 50 μg/ml streptomycin andmethotrexate (100 μM to 200 μM).

b) Principles of the Technique of Molecular Electrophysiology or PatchClamping:

The electrical activity of cells is controlled by the presence andfunctioning of transmembrane pores, the ion channels. The ion channelsare proteins, the conformational state of which can be modified inresponse to various factors: the transmembrane electrical field, thebinding of ligands or post-transcriptional biochemical reactions. Thecurrent passing through an ion channel is of the order of one billionthof an Ampère (pica-Ampère, 1 pA=10⁻¹² A). It can be measured bytechniques of molecular electrophysiology, more commonly called patchclamping.

With the conventional techniques of measuring transmembrane currents byintracellular microelectrodes, the thermodynamic base noise is at leasta hundred times greater than the current passing through a single ionchannel. Under such conditions, the flow in a channel is masked by thisnoise, the variation of which increases with the mean current. ErwinNeher and Bert Sackman (see Hamill et al., 1981) of the Max PlanckInstitute of Góttingen have shown that by analysis of this noise, it waspossible to estimate the current passing in an ion channel. Thethermodynamic base noise is proportional to the area of the membrane. Bylimiting this, the base noise thus becomes lower than the currentpassing through the channel.

The patch clamping technique is derived from the observation made bythese two researchers and their colleagues: a glass micropipette appliedto a membrane surface adheres there such that the electrical resistanceestablished between the pipette and the membrane reaches a gigaohm value(1 Gohm=10⁹ ohm). Ohm's law gives the electrical resistance (R) withrespect to the current intensity I (in Amperes) and the potential U (involts) applied (equation 1) and enables the unit conductance (unit: thepicoSiemens, ps) of the channel g to be determined (equation 2).

U=R.I  (equation 1)

g=1/R  (equation 2)

The interaction forces between the glass of the pipette and thephospholipids of the membrane enable the leakage currents to be reduced,an essential stage for measuring the current passing through an ionchannel. The configuration obtained in this way is designated by theterm “cell-attached”. By withdrawing the pipette starting from thecell-attached configuration, a fragment (patch) of the membrane is tornaway, and remains fixed on the end of the pipette. The configurationobtained in this way is called “inside-out”, since the intracellularsurface of the membrane is in the bath. The extracellular surface of themembrane is in contact with the solution contained in the pipette, whileintracellular part is in contact with the perfusion medium of theexperimental chamber. An electronic assembly allows application(clamping) of a potential difference (Vref−Vp) between the referenceelectrode of the bath (Vref) and the pipette (Vp) and measurement of theresultant current I. If the ionic composition is the same on both sidesof the membrane (symmetric media), the current intensity I is thendirectly proportional to the potential difference applied to themembrane. The points I(V) are generally distributed along a straightline, the gradient and position of which define the unit conductance (g)of the channel and the reversal potential of the current, Erev. At thereversal potential, the flow of charges through the membrane is zero.The reversal potential will be defined by the Nernst equation (equation3), where E represents the Nernst potential for the ion underconsideration and C represents the concentration of the ion in theextracellular (Co) and intracellular (Ci) compartments.

E=RT/zF Log Co/Ci  (equation 3)

In the cell-attached configuration, the potential of the electrode isadded to that of the membrane, which is about −60 mV. In all the cells,the concentrations of potassium (K⁻) and chlorine (Cl⁻) ions are about150 mM and 10 mM respectively. With a pipette containing 150 mM KCl, theNernst potentials for the K⁺ (E_(K)) and Cl⁻ (E_(Cl)) ions will be closeto zero and −50 mV respectively. The reversal of the chlorine currentwill therefore be obtained in the vicinity of the rest potential, thatis to say without application of potential to the membrane (Vp=0 mV). Onthe other hand, the reversal of a potassium current will be obtained bycancelling the potential of the membrane, thus by depolarizing it by 50to 60 mV. This example illustrates the strategies used to evaluate theionic nature of a channel. The information collected by this techniqueis represented by the fluctuation in an electrical current (ofmillisecond order, ms), which conveys the transitions between thevarious states of conductance of the channel.

c) Patch Clamping Applied to the Study of Epithelial Cells in Culture.

The patch clamping experiments are carried out on confluent cells. Afragment of the glass slide (cell support) is placed in an experimentalchamber (volume 600 μl or 1 ml) on the platen of an inverted microscopefitted with phase contrast illumination (Olympus IMT2). Thecell-attached and inside-out configurations are used (Hamill et al.,1981). The experiments are carried out at room temperature (20-22° C.).The currents are amplified with a LIST EPC 7 amplifier (Darmstadt,Germany) (filter of 3 kHz) with a low-pass filter of 2-5 kHz (6-poleBessel filter) and recorded with a DAT (digital audio tape) afterdigitalization (16 bits) at 44 kHz. The data are then transferred to anOlivetti M28PC computer. The pipettes are made from glass tubes of 1 mmdiameter (Clarke Electromedical Instrument) in two or three stages witha horizontal drawer (Flaming/Brown type, model P-87. Sutter. Inst. Co.USA). The pipettes filled with a solution of 150 mM NaCI have aresistance of between 4 and 12 MΩ. The potentials are expressed as thedifference between the potential of the patch electrode and that of thebath. In the cell-attached configuration, they represent the change inpotential with respect to the rest potential of the cell. The diffusionpotentials are evaluated from the potential of the electrodecorresponding to a zero current (when the channels are closed). They areminimized using an agar bridge which establishes the connection betweenthe bath and the reference electrode (earth) and contains the samesolution as the pipette. The reversal potential of the current and theunit conductance of the channels are obtained from the current-voltageratio (I/V) by linear regression. To determine the current-voltageratio, the ionic current amplitudes are measured form amplitudehistograms. The amplitude histograms are shown as the sum of two or moregaussian distributions, the peaks of which correspond to the open andclosed states of channels present in the electrode. From thesehistograms it is possible to determine: N, the total number of channelspresent in the patch: n, the number of channels simultaneously open(n=0, 1, 2 . . . N): Po, the probability of opening of a channel: and I,the mean intensity of the current in a channel. If the channels presentare of the same type and are assumed to open and close independently ofone another, the probability of having n channels open simultaneously isgiven by the binomial distribution (equation 4), from which theindividual probability Po is deduced (equation 5).

P(n)=(N!/n!(N−n)Po ^(n).(1−Po)^(N-n)  (equation 4)

Po=P(n)/N  (equation 5)

Since the present study relates only to the Cl⁻ channels, an exitcurrent would have to be interpreted as a movement of Cl⁻ ions exitingthe pipette towards the intracellular medium of the cells or towards theperfusion medium. The relative permeability P_(X)/P_(Cl) of anion X⁻with respect to Cl⁻ ions has been used to evaluate the ion selectivityof channels in the inside-out configuration. The Goldman-Hodgkin-Katzequation (equation 6) links the ratio of the permeabilities as afunction of the reversal potential (Erev) obtained experimentally andthe respective concentrations of anions present.

Erev=−RT/F In ([Cl⁻]_(e) +P _(X) /P _(Cl) [X ⁻]_(e)/[Cl⁻]_(i) +P _(X) /P_(Cl) [X ⁻]_(i))  (equation 6)

i and e: intracellular and extracellular ion concentration. R. T and Fhave their usual meaning.

For filling the patch electrodes, the composition of the salinesolutions is (in mM): 150 NaCl, 2 MgCl₂, 10 TES (pH 7.4). The perfusionbath of the cells contains (in mM): 145; NaCl, 4 KCl, 2 MgCl₂, 0.5CaCl₂, 10 TES (pH 7.4).

d) Measurement of Short-circuit Currents in an Ussing Chamber.

The benefit of culture of epithelia in chambers with a permeable base,and in particular digestive epithelia (line HT 29 and it various clones,line T84 or Caco2) has been widely demonstrated in cell biology studies.In this technique, the cells are cultured inside a cup where the base ismade of a membrane of polystyrene perforated with holes, the diameter(between 0.45 and 3 μm) and distribution of which are measuredcarefully. It enables attachment of cells without addition of asupplementary matrix. It is transparent in a medium of which therefractive index is close to that of water, and allows visualexamination of the cell layer. However, there are limitations. Althoughthe low thickness of the membrane limits retention of fluids, it cannotbe ruled out that it may constitute a trap for macromolecules orcomplexes, such a gelosomes. This cup of 5 cm² surface area is placed ina 6-well plate. The attachment and the culture of the cells there areconducted in the traditional manner. A fortnight after the inoculation,the cells form an impervious monolayer. This imperviousness is proved bythe occurrence of an electrical resistance or by non-diffusion ofmacromolecules between the two mucous and serous compartments. Theculture is stable for about ten days, maintaining an identical culturemedium in the two compartments. This culture of epithelia in chamberswith a permeable base is quite useful for studying the nature andregulation of secretions and passages of charged or non-chargedmolecules through the epithelium. Transepithelial transportation willdepend on the nature of the permeases present in the two apical orbasolateral fields on either side of the clamped junction. Theproperties of the epithelium which manifests themselves by passage ofcharged molecules can be easily deduced from the measurement of thetransepithelial potential or of the short-circuit current, lichen themolecules are neutral, labelled molecules are used to follow theirmovements.

e) Principles of the Measurement Method.

In outline, epithelial transportation is the balance of celltransportation and selective diffusion through the junction. Celltransportation results from the activity of pairs of specific permeasessituated on the apical pole and on the basal pole of the cellrespectively (Na⁺ and Na⁺/K⁺ATPase channel. Cl⁻ channel and Na⁺/K⁺/Cl⁻cotransporter, Na/glucose cotransporter and diffusional glucosetransporter . . . ). When the transporter epithelium is impervious, theclamped junction isolates two parts of the membrane which have adifferent potential with respect to the medium. A simple electricalanalogy can be used and they can be considered as circuit elementshaving the potentials Vm (mucous) and Vs (serous) respectively. Thesetwo membranes in series thus have a potential Vt which is the algebraicsum Vm+Vs. In the tissue used. Vt can reach −5 mV under standardconditions. To determine the characteristics of the epithelium, threetypes of measurement can be used.

Measurement by an Open Circuit.

The difference in potential existing on either side of the cell layer ismeasured. At a fixed time, a parametrable current i (μA) is sent intothe circuit, which causes a change in the potential difference ΔVproportional to the resistance of the circuit.

Measurement by a Short-circuit Current.

An adjustable current i which uses the resistance of the tissue tocreate a potential difference which will be added algebraically to thatwhich exists is introduced into the circuit. When the current has thevalue Isc (short-circuit current). Vt is zero: Isc×Rt−Et=0 or Isc=Et/Rt.Under the conditions of Vm−Vs=0 and Vm=Vs, the two mucous and serousmembranes are at the same potential. To determine the resistance, agiven potential difference is applied to the circuit and the resistanceis measured, evaluating the deviation in the current Isc which results.

Measurement by an Applied Voltage.

This is a particular case of the measurement by a short-circuit current,where this is applied to the epithelium to give a zero potential. Underthese conditions, the transepithelial potential is fixed without knowingthe potential of each of the membranes. For this, a potential differenceis superimposed, prolonging the algebraic addition of the current usedfor measuring the resistance. A short time is chosen for theshort-circuit current and a long time for the superimposed potentialdifference. It is clear that for a given resistance, the potentialdifference will depend on the capacity of the apparatus to deliver amaximum current (100 μA; for 500 ohms the potential difference is 50mV), but also on the ability to measure the resultant potentialdifference (I V).

To carry out these measurements, the cups are mounted in a modifiedUssing chamber. We use a “current-voltage clamp” (WPI) control unit,coupled to a pulse generator enabling the intensity/voltage curve to beplotted. The signal collected is digitalized (MacLab) and processedusing Chart software on a Macintosh Apple computer.

f) Properties of the Epithelium Tested.

The epithelium formed by HT29 cells will first be used. The addition ofglucose to the base medium (symmetric saline medium, absence ofstimulator) in the mucous compartment causes an increase in thetransepithelial potential and in the short-circuit current withoutaffecting the resistance. The epithelium functions as a glucose absorberwhich utilizes an Na-dependent glucose transporter on the mucous side,and without doubt a diffusional transporter on the serous side. Thetransportation of sodium associated with glucose causes a potentialdifference, which can be inhibited by phlorizin, while the overall flowof glucose is measured with radiolabelled glucose analogues placed inthe mucous or serous compartments.

The addition of agents which cause an increase in the level of cAMP tothe medium induces an increase in Vt and Isc which is independent of theglucose. It appears to be linked to the establishment of atransepithelial transportation of Cl⁻ (serous towards mucous) andinvolves a basolateral chloride transporter on the serous side and achloride channel on the mucous side. This transportation of Cl⁻ isassociated with transportation of water in the same direction. Theapplication, after an increase in the level cAMIP, of agents which causean increase in the level of intracellular Ca²⁻ causes a new increase inVt and Isc associated with, without doubt, a mucous towards serouspassage of Ca²⁺ and a new serous towards mucous passage of Cl⁻.

g) Measurement of the Flows of Radioactive Tracers Applied to the Studyof Epithelial Cells in Culture.

This technique enables the kinetics of the exit of iodide to bemonitored. The cells are cultured in 12-well plates with a dilution to1/10 after passage. On day 3, the drugs to be tested are dissolved inmedium B (37° C.) according to the required concentration. The wells arewashed twice with 1 ml of medium B NaOH, 0.1% glucose, which is thenreplaced by 1 ml of charging solution for 30 min.

The kinetics of the exit of iodide is determined after the chargingsolution has been removed and the wells have been washed 3 times with1.5 ml of medium B. For this, 1 ml of medium B is left in the well for 1min and collected in a haemolysis tube, to be replaced by 1 ml of freshmedium B. The first minute serves as a control, and the product to betested is added from the second minute. The operation is repeated over10 min and the cells are then detached with 1 ml of NaOH 0.1N SDS 0.1%.The contents of each well are collected in a haemolysis tube afteragitation for 25 min, and the tubes are counted for 2 min in a gammacounter.

B) Results

The study relates to molecules of the benzoquinolizinium familydescribed above. They are tested for their ability to activate the CFTRchannel. Screening of molecules as openers of the CFTR channel wascarried out by measuring their effect on the efflux of radioactiveiodide and on the transmembrane chloride currents (Becq et al., 1993a).These data were supplemented by measurement of the level ofintracellular cyclic AMP (cAMP) and its variations in variousexperimental situations.

Three cell models were used to evaluate the effect of CFTR activation bybenzoquinoliziniums: the Xenopus ovocyte injected with the RNA whichcodes for CFTR (this RNA being called cRNA-CFTR in the following): therecombinant CHO cell which expresses the protein CFTR in a stablemanner, and the human colon cell of the line HT29 which expresses theprotein CFTR constitutively. Since the CFTR channel is mainly regulatedby kinase A proteins stimulated by the level of intracellular cAMP,control experiments used derivatives of cAMP which are capable ofpassing through the cell membrane, and the forskoline activator of theenzyme adenylate cyclase, which leads to the synthesis of cAMP in acell. FIG, 1 shows such an activation obtained with 500 μM c-cpt-AMP(cyclic 8-(4-chlorophenylthio)-adenosine 3′,5′-monophosphate), ananalogue of cAMP which passes through the membrane of cells. Theactivation of the CFTR channel, measured by the efflux of iodide,induces an increase in the amplitude of the efflux of iodide (expressedin % of the cell contents at time t=0), and the rate of exit of iodide(gradient of the cures at the origin).

Control experiments which enable the efficacy of the molecules tested onthe activity of the CFTR channel to be evaluated are the following:

1) Xenopus ovocyte: efflux of radioactive iodide and transmembranechloride current in:

ovocytes injected with cRNA-CFTR (labelled CFTR on FIG. 1B);

the same ovocytes in the presence of activators of the cAMP route(c-ctp-AMP, forskoline);

ovocytes injected with water (labelled “water” on FIG. 1B) instead ofcRNA-CFTR;

the same ovocytes in the presence of the abovementioned activators ofthe cAMP route.

2) CHO cell: efflux of radioactive iodide in

CHO cells which have not been transfected with the protein CFTR(labelled CHO-CFTR (−) on FIG. 2 B) in the presence or absence of theabovementioned activators:

CHO cells transfected with the CFTR chain (labelled CHO-CFTR (+) on FIG.2B) in the presence or absence of the abovementioned activators.

3) HT29 cell: efflux of radioactive iodide in

HT29 cells in the absence of activator (labelled basal on FIG. 3);

cells in the presence of the abovementioned activators (labelled cAMP onFIG. 3).

The presence of a chloride channel activated by the increase in thelevel of intracellular calcium was tested in the presence of the calciumionophor A23187. The effects of the activators of cAMP. A23187 andbenzoquinoliziniums were evaluated under these various experimentalconditions (regarded mutatis mutandis as a control, labelled basal) bytheir ability to promote the efflux of radioactive iodide and toincrease the transmembrane chloride current.

FIGS. 2A and 2B show the CFTR activation by 500 μM c-cpt-AMP in therecombinant CHO cell which expresses CFTR. In the control CHO (CFTR−)cell, c-cpt-AMP has no effect. In the HT29 cell, the CFTR channel can bestimulated by cAMP, as the increase in the amplitude of the iodide fluxin the presence of 500 μM c-cpt-AMP shows (FIG. 3A and B).

Effect of Benzoquinoliziniums on the CFTR Channel Activation in the CHOCell

FIG. 4 shows the effect of the derivative MPB-07 (500 μM) on the effluxof iodide in the CHO (CFTR+) and CHO (CFTR−) cell. The activation of theefflux of iodide in the CHO (CFTR+) cell is comparable in intensity andrate to that induced by forskoline (5 μM), an activator of cAMP, in theCHO (CFTR+) cells (FIG. 5). The results regarding the effects of thederivative MPB-07 on the efflux of iodide in the CHO (CFTR−) and CHO(CFTR+) cells are summarized in the histograms of FIGS. 6 and 7respectively. MPB-07 (D00 μM) also effectively stimulates the efflux ofiodide which forskoline (5 μM) produces on the CHO (CFTR+) cells (FIG.7). On these same cells. A23187 (10 μM) has no effect. In the CHO(CFTR−) cells, forskoline (5 μM). A23187 (10 μM), either separately oradded together, and MPB-07 (500 μM) do not significantly modify thebasal level of the efflux of iodide (FIG. 6).

Effect of Benzoquinoliziniums on CFTR Channel Activation in the HT29Cell

The effect of MPB-07 on the efflux of iodide in the recombinant CHO cellis reproduced in the HT29 epithelial cell. The application of 500 μMc-cpt-AMP (FIG. 8A) or of 500 μM MPB-07 (FIG. 8B) triggers an efflux ofiodide, with a similar rate and amplitude, which is increased withrespect to the basal level (without activator) (FIG. 9).

Effect of Benzoquinoliziniums on CFTR Channel Activation Expressed inthe Xenopus Ovocyte

MPB-07 (500 μM) stimulates the efflux of iodide in the Xenopus ovocyte.This activation (FIG. 10A) is comparable to that obtained by applicationof 500 μM c-cpt-AMP (FIG. 10B) and is significantly different (FIG. 11)to the efflux measured in the stimulated (cAMP and MPB-07) andnon-stimulated (basal) non-injected ovocyte (FIG. 11, water) and in theovocyte which is not stimulated but expresses CFTR (FIG. 10A, effluxlabelled basal CFTR).

Structure-function Study of Benzoquinoliziniums and Correlation With theCFTR Opening

The study was carried out on 16 derivatives of the benzoquinoliziniumnucleus.

Tables 1 and 2 show the chemical structure of compounds of thebenzoquinolizinium family tested here as a CFTR channel activator.

Tables 1 and 2 shows the results relating to the efflux measured in thepresence of various compounds tested on the recombinant CHO (CFTR+)cell. The base compound, phenanthrene (table 2) does not activate theCFTR channel. Two series of molecules were tested: NH₂ series (table 1,MPB-26, MPB-01 to 04; table 2: MPB-24) and OH series (table 1, MPB-05 to08, MPB-27, 29, 30 and 32; table 2: MPB-25). The percentages of CFTRchannel activation are given in the corresponding tables. They show thatthe OH series activates CFTR with percentages of between and 110%. TheNH₂ series is less active (10 to 30%).

In the OH series, the efficacy is the following:

MPB-05, 08, 25, 32<30<29<06<27, 07

From all the compounds studied, it seems that the presence of the OHgrouping in position 6 is the determining factor as regards the abilityto activate the CFTR channel:

9 compounds with OH in position 6: 45% CFTR channel activation

6 compounds with NH, in position 6: 13% CFTR channel activation.

Effect of Benzoquinoliziniums on Intracellular cAMP

FIG. 12 shows the levels of cell cAMP in the recombinant CHO cellmeasured after 5 min in the presence of 5 μM forskoline (activator ofthe enzyme for synthesis of cAMP: adenylate cyclase), 10 μM rolipram (aninhibitor of the enzyme of cAMP degradation: type IV phosphodiesterases)and 500 μM MPB-07. The same level of cAMP is reached in the presence ofthe compounds MPB-07 and rolipram, but only MPB-07 triggers CFTR channelactivation. The effect of forskoline on cAMP is multiplied a factor ofabout 4, suggesting that this effect is purely dependent on cAMP,Rolipram has no effect as a CFTR activator measured by the flow ofiodide and by patch clamping. These results show that the compoundMPB-07 stimulates the CFTR channel by a route independent of the cellcAMP route.

C) Conclusion

These results show that the compound MPB-07 and some members of thebenzoquinolizinium family stimulate opening of the CFTR channel by aroute independent of cAMP or intracellular calcium. These molecules thusrepresent a new family of CFTR channel activators.

LEGENDS TO FIGURES

FIG. 1: Effect of c-cpt-AMP on the efflux of radioactive iodide in theXenopus ovocyte;

FIG. 1A: Curves of the efflux of ¹²⁵I (% on the ordinate) as a functionof time (min on the abscissa): the curve passing through pointsrepresented by black squares corresponds to the efflux of ¹²⁵I measuredas a function of time in ovocytes injected with cRNA-CFTR (curveddesignated basal CFTR): the curve passing through points represented bywhite square corresponds to the efflux of ¹²⁵I measured as a function oftime in ovocytes injected with cRNA-CFTR and activated by c-cpt-AMP(curve designated CFTR+cAMP);

FIG. 1B: Histograms of the efflux of ¹²⁵I in ovocytes which have notbeen activated by c-cpt-AMP (shown in black) and in ovocytes activatedby c-cpt-AMP (shown in white): ovocytes which have or have not beenactivated by c-cpt-AMP and have been injected with water are shown onthe left (labelled “water”): ovocytes which have or have not beenactivated by c-cpt-AIMP and have been injected with cRNA-CFTR are shownon the right (labelled “CFTR”): n represents the number of experiments.

FIG. 2: Effect of c-cpt-AMP on the efflux of radioactive iodide in theCHO cells:

FIG. 2A: Curves of the efflux of ¹²⁵I (% on the ordinate) as a functionof time (min on the abscise): the curve passing through pointsrepresented by black triangles corresponds to the efflux of ¹²⁵Imeasured as a function of time in the CHO cells which have not beenactivated by c-cpt-AMP (curve designated basal); the curve passingthrough points represented by white triangles corresponds to the effluxof ¹²³I measured as a function of time in the CHO cells activated byc-cpt-AMP (curve designated cAMP);

FIG. 2B: Histograms of the efflux of ¹²⁵I in the CHO cells which havenot been activated by c-cpt-AMP (shown in white); the CHO cells whichhave or have not been activated by c-cpt-AMP and have not beentransfected with the CFTR gene are shown on the left (labelled CHO(CFTR−)); the CHO cells which have or have not been activated byc-cpt-AMP and have been transfected with the CFTR gene are shown on theright (labelled CHO (CFTR+)); n represents the number of experiments.

FIG. 3: Effect of c-cpt-AMP on the efflux of radioactive iodide in HT29cells:

FIG. 3A: Curves of the efflux of ¹²⁵I (% on the ordinate) as a functionof time (min on the abscise): the curve passing through pointsrepresented by black squares corresponds to the efflux of ¹²⁵I measuredas a function of time in HT29 cells which have not been activated byc-cpt-AMP (curve designated basal): the curve passing through pointsrepresented by white squares corresponds to the efflux of ¹²⁵I measuredas a function of time in the HT29 cells activated by c-cpt-AMP (curvedesignated cAMP):

FIG. 3B: Histograms of the efflux of ¹²⁵I in the HT29 cells which havenot been activated b c-cpt-AMP (in black) and in the HT29 cellsactivated by c-cpt-AMP (in white).

FIG. 4: Effect of the derivative MPB-07 (500 μM) on the efflux of ¹²⁵I(% on the ordinate) as a function of time (min on the abscise) in theCHO cell: the curve passing through points represented by white circlescorresponds to the measurement of the efflux of ¹²⁵I as a function oftime in the CHO cells activated by MPB-07 and transfected with the CFTRgene (curve designated MPB-07 (CFTR+)): the curve passing through pointsrepresented by black circles corresponds to the measurement of theefflux of ¹²⁵I as a function of time in the CHO cells activated byMPB-07 but not transfected with the CFTR gene (curve designated MPB-07(CFTR−)).

FIG. 5: Effect of “forskoline” (500 μM) on the efflux of ¹²⁵I (% on theordinate) as a function of time (min on the abscise) in the CHO cell;the curve passing through points represented by white trianglescorresponds to the measurement of the efflux of ¹²⁵I as a function timein CHO cells activated by forskoline and transfected with the CFTR gene(curve designated forskoline CHO (CFTR+)); the curve passing throughpoints represented by black triangles corresponds to the measurement ofthe efflux of ¹²⁵I as a function of time in the CHO cells which have notbeen activated by forskoline and have been transfected with the CFTRgene (curve designated basal CHO (CFTR+)).

FIG. 6: Histograms of the efflux of 125I in the CHO cells which have notbeen transfected with the CFTR gene (designated CHO (CFTR−)) and:

have not been activated (basal)

have been activated by forskoline (forskoline)

have been activated by A23187 (A23187)

have been activated by A23187 and forskoline (A23187+fsk)

have been activated by MPB-07 (MPB-07)

n represents the number of experiments.

FIG. 7: Histograms of the efflux of ¹²⁵I in the CHO cells which havebeen transfected with the CFTR gene (designated CHO (CFTR+)) and:

have not been activated (basal)

have been activated by forskoline (forskoline)

have been activated by MPB-07 (MPB-07)

have been activated by A23187 (A23187)

n represents the number of experiments.

FIG. 8: Comparison of the effects of c-cpt-AMP (500 μM) and MPB-07 (500μM) on the efflux of ¹²⁵I (% on the ordinate) as a function of time (minon the abscise) in the HT29 cells;

FIG. 8A: The curve passing through points represented by black squarescorresponds to the measurement of the efflux of 125I as a function oftime in the non-activated HT29 cells (curve designated basal): the curvepassing through points represented by white squares corresponds to themeasurement of the efflux of ¹²⁵I as a function of time in the HT29cells activated by c-ctp-AMP (curve designated cAMP):

FIG. 8B: The curve passing through points represented by white squarescorresponds to the measurement of the efflux of ¹²⁵I as a function oftime in the non-activated HT29 cells (curve designated basal): the curvepassing through points represented by black squares corresponds to themeasurement of the efflux of ¹²⁵I as a function of time in the HT29cells activated by MPB-07 (curve designated MPB-07).

FIG. 9: Histogram of the efflux of 125I in the HT29 cells which have notbeen activated (basal), have been activated by c-ctp-AMP (cAMP) and havebeen activated by MPB-07 (MPB-07): n represents the number ofexperiments.

FIG. 10: Comparison of the effects of c-ctp-AMP (500 μM) on the effluxof ¹²⁵I (% on the ordinate) as a function of time (min on the abscise)in the Xenopus ovocytes;

FIG. 10A: The curve passing through points represented by white circlescorresponds to the measurement of the efflux of 125I as a function oftime in the ovocytes injected with water (curve designated basal water):the curve passing through points represented by black squarescorresponds to the measurement of the efflux of ¹²⁵I as a function oftime in the ovocytes injected with cRNA-CFTR (curve designated basalCFTR): the curve passing through points represented by white squarescorresponds to the measurement of the efflux of H231 as a function oftime in the ovocytes which have been injected with cRNA-CFTR and havebeen activated with c-ctp-AMP (curve designated CFTR+cAMP);

FIG. 10B: The curve passing through points represented by white squarescorresponds to the measurement of the efflux of 125I as a function oftime in the ovocytes which have been injected with cRNA-CFTR and haveactivated by MPB-07 (curve designated MPB-07).

FIG. 11: Histograms of the efflux of 125I in the Xenopus ovocytes whichhave not been activated (basal), or have been activated by c-ctp-AMP(cAMP), or have been activated by MPB-07 (MPB-07), these ovocytes havingbeen either injected with water (labelled “water” on the left), orinjected with cRNA-CFTR (labelled “CFTR” on the right); n represents thenumber of experiments.

FIG. 12: Histograms representing the levels of cAMP (measured in nmolper mg of protein) in the CHO cells which have been transfected with theCFTR gene and have not been activated (basal), or have been activated byforskoline (forskoline), or have been activated by rolipram (rolipram),or have been activated by MPB-07 (MPB-07).

TABLE 1

% activation relative Compound X Y R to control MPB-26 Cl NH₂ H 30MPB-03 Cl NH₂ 9-Cl 18 MPB-04 Cl NH₂ 7Cl 11 MPB-06 Br OH H 53 MPB-05 ClOH H 20 MPB-07 Cl OH 10-Cl 110  MPB-08 Cl OH 9-Cl 22 MPB-27 Cl OH 7-Cl95 MPB-30 Cl OH 8-Cl 28 MPB-29 Cl OH 9-F 42 MPB-32 Cl OH 8-Br 13 control(basal)  0 forskoline 109 

N.B.: forskoline: 5 μM, MPB et phenanthrene 500 μM.

TABLE 2

% activation relative Compound X Y R to control phenanthrene H H H <5MPB-24 H NH₂ H 18 MPB-25 H OH H 25

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What is claimed is:
 1. Compositions for activating cystic fibrosistransmembrane conductance regulator channels in vivo, said compositionscontaining at least one benzo[c]quinolizinium derivative selected fromthe group consisting of:

in combination with a physiologically acceptable vehicle.